8 Microglial subpopulations
Identification of Microglial Subpopulations after reclustering
8.1 Reclustered microglia
The integrated scRNAseq dataset of all brain cell populations was subsetted to retain only cells annotated as microglia by the “expert committee”. The subset was reprocessed using the assay integrated to ensure batch-corrected expression values were used for downstream clustering.
Dimensionality reduction was performed by principal component analysis (PCA) on the set of variable features. An elbow plot was generated to identify the inflection point, and the first 10 principal components were selected for neighborhood graph construction.
Nearest neighbors in the reduced space were computed and an unsupervised clustering across a range of resolutions (0.1 to 1.0, step 0.2) was performed. Cluster stability across resolutions was evaluated with the clustree package, and a resolution of 0.3 was selected as a balance between capturing subpopulation diversity and avoiding overfragmentation.
The final clusters were visualized using UMAP. The reclustering identified seven transcriptionally distinct microglial subpopulations (clusters 0–6) (Fig. 1B), compared with the initial single microglia cluster in the whole-brain dataset (Fig. 1A). These clusters exhibited clear separation in UMAP space, indicating distinct transcriptional programs.
| 0 | 1 | 2 | 3 | 5 | 8 | 12 | 13 | 14 | 28 | |
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 6007 | 146 | 101 | 95 | 0 | 189 | 14 | 0 | 3 | 45 |
| 1 | 25 | 4829 | 1 | 283 | 40 | 101 | 42 | 795 | 6 | 3 |
| 2 | 196 | 121 | 4783 | 3 | 78 | 45 | 41 | 0 | 3 | 65 |
| 3 | 47 | 31 | 1 | 3337 | 7 | 2 | 709 | 15 | 11 | 15 |
| 4 | 0 | 13 | 8 | 1 | 3397 | 0 | 0 | 188 | 6 | 1 |
| 5 | 603 | 18 | 14 | 17 | 1 | 2144 | 527 | 18 | 3 | 8 |
| 6 | 14 | 83 | 3 | 1 | 25 | 0 | 6 | 1 | 944 | 4 |
8.2 Cluster markers
Cluster-specific marker genes were determined using a Wilcoxon rank-sum test, restricted to genes expressed in at least 30% of cells within a cluster.
The table represents the top 20 markers (according to the adjusted p-value) for each cluster:
| cluster | top_markers |
|---|---|
| 0 | Nfkbia (Upregulated) , Gadd45b (Upregulated) , Tnf (Upregulated) , Egr1 (Upregulated) , Nfkbiz (Upregulated) , Ier2 (Upregulated) , Ier3 (Upregulated) , Il1b (Upregulated) , Bcl2a1d (Upregulated) , Tnfaip3 (Downregulated), Ccl4 (Upregulated) , Mir155hg (Upregulated) , Nr4a3 (Upregulated) , Junb (Upregulated) , Ifrd1 (Upregulated) , Btg2 (Downregulated) , Clic4 (Upregulated) , Fos (Downregulated) , Ccrl2 (Upregulated) , Ccl3 (Upregulated) |
| 1 | Zfp36 (Downregulated) , Btg2 (Downregulated) , Cd83 (Downregulated) , Dennd4a (Downregulated) , Atf3 (Downregulated) , Rel (Downregulated) , Ier5 (Downregulated) , Jun (Downregulated) , H3f3b (Downregulated) , Mapkapk2 (Downregulated), Baiap2l2 (Upregulated) , Cebpb (Downregulated) , Zfp36l1 (Downregulated) , Or5v1b (Upregulated) , Tmem163 (Upregulated) , Btg1 (Downregulated) , Plek (Downregulated) , Serpine2 (Upregulated) , Cst7 (Downregulated) , Ank (Downregulated) |
| 2 | Ctsb (Downregulated) , Serpine2 (Downregulated), Tgm2 (Upregulated) , Cacnb2 (Upregulated) , Klf2 (Upregulated) , Il1a (Upregulated) , Socs3 (Upregulated) , Tmem119 (Upregulated) , Trem2 (Downregulated) , Cd68 (Downregulated) , Syngr1 (Downregulated) , Fos (Upregulated) , Ccr5 (Upregulated) , Crybb1 (Upregulated) , Nr4a1 (Upregulated) , Jun (Upregulated) , Rapgef5 (Upregulated) , Ctsd (Downregulated) , Jund (Upregulated) , Rnf19b (Upregulated) |
| 3 | Spp1 (Upregulated) , Atp6v0d2 (Upregulated) , H2-Aa (Upregulated) , Gpnmb (Upregulated) , H2-Eb1 (Upregulated) , Itgax (Upregulated) , Fabp5 (Upregulated) , Dkk2 (Upregulated) , Trpc4 (Upregulated) , H2-Ab1 (Upregulated) , Ctnna3 (Upregulated) , Siglech (Downregulated), P2ry12 (Downregulated) , Cst7 (Upregulated) , Igf1 (Upregulated) , Plau (Upregulated) , Cd74 (Upregulated) , Mamdc2 (Upregulated) , Lpcat2 (Downregulated) , Lgals3 (Upregulated) |
| 4 | Cacnb2 (Upregulated) , Ctsb (Downregulated) , Plek (Downregulated) , Bank1 (Upregulated) , Gm10790 (Upregulated) , Tyrobp (Downregulated) , Frmd4a (Upregulated) , Tmem119 (Upregulated) , Ccr5 (Upregulated) , Rapgef5 (Upregulated) , Maf (Upregulated) , Cd9 (Downregulated) , Trem2 (Downregulated) , A830008E24Rik (Upregulated), Lyz2 (Downregulated) , Cd68 (Downregulated) , Ctsz (Downregulated) , Csmd3 (Upregulated) , Syngr1 (Downregulated) , Ifitm10 (Upregulated) |
| 5 | Itgam (Downregulated) , Ifi207 (Upregulated) , C1qb (Downregulated) , Ccl4 (Upregulated) , Lilrb4a (Upregulated) , Ly86 (Upregulated) , C1qc (Downregulated) , Fgf13 (Upregulated) , C1qa (Downregulated) , Klf6 (Upregulated) , Dusp1 (Upregulated) , Atf3 (Upregulated) , P2ry12 (Downregulated), Marcks (Downregulated), Rgs10 (Downregulated) , Cst3 (Downregulated) , Ctss (Downregulated) , Hexb (Downregulated) , Laptm5 (Downregulated), Xylt1 (Upregulated) |
| 6 | Ifit3 (Upregulated) , Ifi211 (Upregulated) , Ifi206 (Upregulated) , Ifit2 (Upregulated) , Rsad2 (Upregulated) , Isg15 (Upregulated) , Mx1 (Upregulated) , Ifit3b (Upregulated) , Ifi213 (Upregulated) , Oasl2 (Upregulated) , A330040F15Rik (Upregulated), Ifi209 (Upregulated) , Slfn5 (Upregulated) , Gm4951 (Upregulated) , Ifitm3 (Upregulated) , Stat1 (Upregulated) , Ifi207 (Upregulated) , Rnf213 (Upregulated) , Sp100 (Upregulated) , Herc6 (Upregulated) |
8.3 Cluster interpretation (chatGPT 5)
8.3.1 Cluster 0
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Nfkbia | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | NF-κB inhibitor; rapid-response gene in inflammatory microglia during cytokine signaling. |
| Gadd45b | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Stress-responsive transcriptional regulator induced by pro-inflammatory stimuli in microglia. |
| Tnf | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Classic pro-inflammatory cytokine, hallmark of microglial activation. |
| Egr1 | Microglia | Immediate early gene (IEG)-activated microglia | Upregulated | Rapid transcriptional activator in microglia after acute stimuli; linked to inflammatory and stress responses. |
| Nfkbiz | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | NF-κB pathway regulator; modulates pro-inflammatory gene expression in microglia. |
| Ier2 | Microglia | Immediate early gene (IEG)-activated microglia | Upregulated | Early-response transcription factor upregulated in activated microglia after stimulation. |
| Ier3 | Microglia | Immediate early gene (IEG)-activated microglia | Upregulated | Anti-apoptotic and stress response mediator; induced in activated microglia. |
| Il1b | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Key pro-inflammatory cytokine secreted by activated microglia; promotes neuroinflammation. |
| Bcl2a1d | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Anti-apoptotic protein induced by NF-κB in inflammatory microglia. |
| Tnfaip3 | Microglia | Activated/inflammatory microglia (AIM) | Downregulated | Negative regulator of NF-κB signaling; lower levels can prolong inflammatory responses. |
| Ccl4 | Microglia | Chemotactic/inflammatory microglia | Upregulated | Chemokine attracting immune cells; enriched in DAM/AIM microglia. |
| Mir155hg | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Host gene for miR-155, a pro-inflammatory regulator in microglia. |
| Nr4a3 | Microglia | Immediate early gene (IEG)-activated microglia | Upregulated | Nuclear receptor involved in transcriptional reprogramming after activation. |
| Junb | Microglia | Immediate early gene (IEG)-activated microglia | Upregulated | Component of AP-1 transcription factor complex; induced by inflammatory and stress signals. |
| Ifrd1 | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Transcriptional regulator linked to immune cell activation. |
| Btg2 | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Early-response gene; reduction may reflect shift from transient to sustained activation. |
| Clic4 | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Chloride channel involved in microglial activation and phagocytosis. |
| Fos | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Classic IEG; lower levels may indicate post-acute or chronic activation phase. |
| Ccrl2 | Microglia | Chemotactic/inflammatory microglia | Upregulated | Chemokine receptor-like protein promoting immune cell recruitment during neuroinflammation. |
| Ccl3 | Microglia | Chemotactic/inflammatory microglia | Upregulated | Pro-inflammatory chemokine; signature of activated and DAM microglia. |
All 20 genes are microglial, with the majority linked to activated/inflammatory microglia (AIM) and chemotactic responses, indicating strong pro-inflammatory activation. A large subset are immediate early genes (Egr1, Ier2, Ier3, Nr4a3, Junb, Fos, Btg2), suggesting acute transcriptional reprogramming. Several chemokines (Ccl3, Ccl4) and receptors (Ccrl2) point to immune cell recruitment, while NF-κB pathway components (Nfkbia, Nfkbiz, Tnfaip3) highlight central inflammatory signaling. The pattern is consistent with a robust, possibly sustained inflammatory microglial state with recruitment and signaling functions active.
8.3.2 Cluster 1
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Zfp36 | Microglia | Homeostatic microglia | Downregulated | RNA-binding protein promoting resolution of inflammation; reduced expression suggests loss of anti-inflammatory tone. |
| Btg2 | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Early-response regulator; decreased levels may indicate reduced acute transcriptional activation. |
| Cd83 | Microglia | Activated/antigen-presenting microglia | Downregulated | Co-stimulatory molecule; lower levels suggest dampened antigen presentation capacity. |
| Dennd4a | Microglia | Homeostatic microglia | Downregulated | Endosomal trafficking regulator; reduced expression may impair normal vesicular function. |
| Atf3 | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Stress-responsive transcription factor; decrease suggests reduced acute stress response. |
| Rel | Microglia | Activated/inflammatory microglia (AIM) | Downregulated | NF-κB subunit; downregulation indicates suppressed inflammatory transcription program. |
| Ier5 | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Early-response transcriptional regulator; lower levels indicate reduced activation. |
| Jun | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | AP-1 complex member; decreased expression points to reduced stimulus-driven gene expression. |
| H3f3b | Microglia | Homeostatic microglia | Downregulated | Histone variant linked to active transcription; reduction may reflect lower transcriptional turnover. |
| Mapkapk2 | Microglia | Activated/inflammatory microglia (AIM) | Downregulated | p38 MAPK pathway kinase; downregulation suggests decreased cytokine production signaling. |
| Baiap2l2 | Microglia | Potentially reactive microglia | Upregulated | Actin cytoskeleton regulator; increased levels may support morphological remodeling. |
| Cebpb | Microglia | Activated/inflammatory microglia (AIM) | Downregulated | Transcription factor driving inflammatory and DAM programs; reduced levels suggest attenuated activation. |
| Zfp36l1 | Microglia | Homeostatic microglia | Downregulated | Post-transcriptional regulator of inflammatory mRNAs; decrease may affect RNA stability control. |
| Or5v1b | Microglia | Uncharacterized/possible off-target expression | Upregulated | Olfactory receptor-like; rare in microglia, upregulation could reflect atypical activation. |
| Tmem163 | Microglia | Homeostatic microglia | Upregulated | Vesicular zinc transporter; increase may relate to altered ion handling in reactive states. |
| Btg1 | Microglia | Immediate early gene (IEG)-activated microglia | Downregulated | Cell cycle regulator with early-response profile; reduction aligns with quiescence. |
| Plek | Microglia | Homeostatic microglia | Downregulated | Cytoskeletal-associated protein; decrease may reflect reduced motility or phagocytic activity. |
| Serpine2 | Microglia | Remodeling/reactive microglia | Upregulated | Extracellular matrix regulator; elevated in tissue remodeling and repair contexts. |
| Cst7 | Microglia | Activated/DAM microglia | Downregulated | Cysteine protease inhibitor; lower expression may impair control of proteolytic activity. |
| Ank | Microglia | Homeostatic microglia | Downregulated | Cytoskeletal anchoring protein; decrease may indicate reduced stability of cell processes. |
Most genes in cluster 1 are downregulated and associated with homeostatic, immediate early, or inflammatory microglial programs, indicating an overall suppression of activation and immune signaling. Notably, transcription factors (Rel, Jun, Cebpb) and RNA regulators (Zfp36, Zfp36l1) are decreased, suggesting reduced inflammatory transcription and mRNA turnover. A few genes (Baiap2l2, Tmem163, Serpine2) are upregulated, hinting at selective morphological remodeling and extracellular matrix modulation. Overall, the profile suggests a shift toward a less inflammatory, potentially partially remodeling/reactive microglial state.
8.3.3 Cluster 2
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Ctsb | Microglia | Lysosomal/phagocytic (DAM-like) | Downregulated | Cathepsin B; reduced levels suggest diminished phagolysosomal activity typical of DAM/phagocytic programs. |
| Serpine2 | Microglia | Remodeling/reactive | Downregulated | ECM serine protease inhibitor; decrease implies less extracellular remodeling. |
| Tgm2 | Microglia | Activated/reactive microglia | Upregulated | Transglutaminase 2; induced in activated microglia, supports adhesion/phagocytosis and Aβ handling. |
| Cacnb2 | Microglia | Reactive signaling microglia | Upregulated | Ca²⁺ channel β subunit; heightened Ca²⁺ signaling aligns with reactive state transitions. |
| Klf2 | Microglia | Homeostatic/anti‑inflammatory | Upregulated | Transcription factor that restrains inflammatory programs; rise suggests homeostatic pushback. |
| Il1a | Microglia | Activated/inflammatory microglia (AIM) | Upregulated | Pro‑inflammatory cytokine driving local neuroinflammation. |
| Socs3 | Microglia | Activated (JAK/STAT feedback) | Upregulated | Negative feedback regulator of cytokine signaling; elevation marks acute activation with braking. |
| Tmem119 | Microglia | Homeostatic microglia | Upregulated | Core microglial identity marker; increase indicates preserved homeostatic signature. |
| Trem2 | Microglia | DAM/phagocytic | Downregulated | Key receptor for DAM induction and lipid/Aβ phagocytosis; drop argues against DAM engagement. |
| Cd68 | Microglia | Phagocytic/activated | Downregulated | Lysosomal/phagolysosomal marker; reduction indicates lower phagocytic tone. |
| Syngr1 | Microglia | Contamination/neuronal signal | Downregulated | Synaptic vesicle protein (neuronal); decreased expression fits reduced ambient neuronal RNA. |
| Fos | Microglia | Immediate‑early gene (IEG)‑activated | Upregulated | Classic IEG; marks acute transcriptional activation. |
| Ccr5 | Microglia | Chemotactic/inflammatory | Upregulated | Chemokine receptor; suggests migration/recruitment during inflammatory response. |
| Crybb1 | Microglia | Stress‑responsive/reactive | Upregulated | Crystallin family chaperone; induced under cellular stress in glia. |
| Nr4a1 | Microglia | IEG/anti‑inflammatory reprogramming | Upregulated | Nuclear receptor shaping rapid activation and tolerance programs. |
| Jun | Microglia | IEG‑activated | Upregulated | AP‑1 component; hallmark of acute stimulus response. |
| Rapgef5 | Microglia | Motility/chemotaxis signaling | Upregulated | Rap1 GEF; supports cytoskeletal dynamics and process motility in reactive microglia. |
| Ctsd | Microglia | Lysosomal/phagocytic (DAM-like) | Downregulated | Cathepsin D; decrease reinforces reduced lysosomal/phagocytic program. |
| Jund | Microglia | IEG‑activated | Upregulated | AP‑1 family factor; marks early activation. |
| Rnf19b | Microglia | Stress/adaptive (proteostasis) | Upregulated | E3 ubiquitin ligase (ERAD/quality control); elevation indicates proteostatic stress response. |
Cluster 2 combines strong IEG/cytokine activation (Fos/Jun/Jund, Il1a, Socs3, Ccr5) with preservation of homeostatic identity (Tmem119, Klf2↑) and suppression of DAM/phagolysosomal genes (Trem2, Cd68, Ctsb, Ctsd↓). This pattern supports an acute, non‑phagocytic inflammatory microglial state with chemotactic/migratory bias and active negative‑feedback (Socs3), rather than a classic Trem2⁺ DAM phenotype. Tgm2 and stress markers (Crybb1, Rnf19b) suggest matrix/adhesion changes and proteostatic adaptation during this transient activation.
8.3.4 Cluster 3:
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Spp1 | Microglia | Spp1⁺ DAM (osteopontin-high) | Upregulated | Canonical Spp1⁺ neurodegeneration-associated subset; linked to phagolysosomal activation and plaque-proximal microglia. |
| Atp6v0d2 | Microglia | Spp1⁺ DAM | Upregulated | Lysosomal/osteoclast-like proton pump subunit enriched in Spp1⁺ DAM; supports catabolic/phagolysosomal programs. |
| H2-Aa | Microglia | MHC-II⁺ antigen-presenting microglia (ARM) | Upregulated | MHC-II component indicating antigen presentation. |
| Gpnmb | Microglia | DAM/MGnD | Upregulated | Injury/DAM marker enriched in neurodegenerative microglia and lipid-handling states. |
| H2-Eb1 | Microglia | MHC-II⁺ ARM | Upregulated | MHC-II beta-chain; antigen presentation program. |
| Itgax | Microglia | DAM/MGnD (CD11c⁺) | Upregulated | CD11c⁺ “activated”/MGnD microglia; plaque-associated. |
| Fabp5 | Microglia | Lipid-handling DAM / LDAM-like | Upregulated | Fatty-acid–binding protein; lipid metabolism and phagocytosis in activated microglia. |
| Dkk2 | Microglia | Neurodegenerative-DAM subset | Upregulated | Wnt antagonist selectively induced in mouse neurodegenerative microglia near plaques. |
| Trpc4 | Microglia | Activated/DAM-associated | Upregulated | Ca²⁺ channel up in activated microglia; supports motility/chemotaxis in DAM contexts. |
| H2-Ab1 | Microglia | MHC-II⁺ ARM | Upregulated | MHC-II alpha-chain; part of antigen-presenting signature. |
| Ctnna3 | Microglia | Activated/DAM-associated | Upregulated | Cell-adhesion gene reported in activated/DAM-like microglia; may reflect contact with dystrophic neurites. |
| Siglech | Microglia | Homeostatic | Downregulated | Homeostatic microglia marker; its loss marks transition to non-homeostatic/DAM states. |
| P2ry12 | Microglia | Homeostatic | Downregulated | Core homeostatic microglia receptor; down in activated/DAM microglia. |
| Cst7 | Microglia | DAM/MGnD | Upregulated | Cathepsin inhibitor robustly induced in DAM; linked to lysosomal remodeling. |
| Igf1 | Microglia | IGF1⁺ reparative microglia | Upregulated | Trophic/reparative program; promotes oligodendrocyte/myelin support within disease context. |
| Plau | Microglia | Activated ECM-remodeling/DAM | Upregulated | uPA; extracellular matrix remodeling and migration; increased in activated plaque-associated microglia. |
| Cd74 | Microglia | MHC-II⁺ ARM | Upregulated | Invariant chain for MHC-II; hallmark of antigen-presenting microglia. |
| Mamdc2 | Microglia | Activated/DAM-associated | Upregulated | Extracellular/adhesion protein reported in activated plaque-proximal microglia subsets. |
| Lpcat2 | Microglia | Pro‑inflammatory eicosanoid program | Downregulated | Arachidonoyl-lysophosphatidylcholine acyltransferase; lower levels suggest shift away from classical lipid‑inflammatory signaling. |
| Lgals3 | Microglia | Activated/DAM (Galectin‑3⁺) | Upregulated | Galectin‑3 drives phagocytosis and TREM2 signaling; strong DAM/plaqued‑associated marker. |
Most markers map to disease-associated microglia (DAM), including a prominent Spp1⁺/osteoclast‑like module (Spp1, Atp6v0d2, Gpnmb, Fabp5, Dkk2, Itgax, Cst7, Lgals3) and an antigen-presenting MHC‑II arm (H2-Aa/H2-Ab1/H2-Eb1, Cd74). Homeostatic signatures (P2ry12, Siglech) are down, consistent with an activated, plaque‑proximal state. Additional genes (Plau, Trpc4, Ctnna3, Mamdc2) support activation/motility and ECM remodeling, while Igf1 suggests a concurrent reparative program. Overall, cluster 3 reflects plaque‑associated, antigen‑presenting Spp1⁺ DAM with suppressed homeostatic identity.
8.3.5 Cluster 4
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Cacnb2 | Microglia | Reactive signaling microglia | Upregulated | Voltage-gated Ca²⁺ channel β subunit; promotes altered Ca²⁺ dynamics during activation and migration. |
| Ctsb | Microglia | Lysosomal/phagocytic (DAM-like) | Downregulated | Cathepsin B; decrease suggests reduced lysosomal proteolysis and phagocytic capacity. |
| Plek | Microglia | Homeostatic microglia | Downregulated | Cytoskeletal-associated protein; reduced levels may impair motility and process extension. |
| Bank1 | Microglia | Immune signaling–biased microglia | Upregulated | Scaffold protein in B/TLR signaling; induction suggests immune receptor pathway engagement. |
| Gm10790 | Microglia | Uncharacterized/reactive | Upregulated | Predicted gene with reported glial expression in activation contexts. |
| Tyrobp | Microglia | DAM/phagocytic | Downregulated | Adaptor for TREM2/Syk signaling; lower levels indicate reduced DAM engagement. |
| Frmd4a | Microglia | Reactive/motility remodeling | Upregulated | Actin cytoskeleton regulator; supports polarity and migration. |
| Tmem119 | Microglia | Homeostatic microglia | Upregulated | Core homeostatic marker; maintenance suggests preserved identity amid activation. |
| Ccr5 | Microglia | Chemotactic/inflammatory | Upregulated | Chemokine receptor driving migration toward inflammatory cues. |
| Rapgef5 | Microglia | Motility/chemotaxis signaling | Upregulated | Regulates Rap1 activity and cytoskeletal rearrangement in reactive microglia. |
| Maf | Microglia | Anti-inflammatory/regulatory | Upregulated | Transcription factor promoting repair/tolerogenic states; elevated in certain homeostatic-like contexts. |
| Cd9 | Microglia | DAM/phagocytic | Downregulated | Tetraspanin in exosome/lysosome biology; decrease indicates reduced vesicular/phagocytic functions. |
| Trem2 | Microglia | DAM/phagocytic | Downregulated | Receptor essential for DAM induction; lower expression suggests non-DAM inflammatory state. |
| A830008E24Rik | Microglia | Uncharacterized/reactive | Upregulated | Predicted transcript enriched in activated glia. |
| Lyz2 | Microglia | DAM/phagocytic | Downregulated | Lysozyme C2; loss indicates reduced microbicidal/lysosomal activity. |
| Cd68 | Microglia | Lysosomal/phagocytic | Downregulated | Lysosomal membrane glycoprotein; decline suggests reduced phagolysosomal activity. |
| Ctsz | Microglia | Lysosomal/phagocytic | Downregulated | Cathepsin Z; decrease aligns with downregulated phagolysosomal program. |
| Csmd3 | Microglia | Reactive/adhesion-modulating | Upregulated | Large complement regulatory protein; linked to cell–cell/ECM interactions. |
| Syngr1 | Microglia | Contamination/neuronal signal | Downregulated | Synaptic vesicle protein; likely ambient neuronal transcript reduced in this cluster. |
| Ifitm10 | Microglia | Stress-adaptive | Upregulated | IFITM family member; up in stress/injury contexts, potential role in membrane remodeling. |
Cluster 4 shows a mixed profile: upregulation of signaling, motility, and immune-modulating genes (Cacnb2, Bank1, Ccr5, Rapgef5, Maf) with concurrent preservation of homeostatic identity (Tmem119↑) and marked downregulation of DAM/phagolysosomal machinery (Trem2, Tyrobp, Cd68, Ctsb, Ctsz, Lyz2). This suggests a non-DAM reactive state biased toward chemotaxis and cytoskeletal remodeling rather than debris clearance. The pattern could reflect microglia mobilized for migration or vascular surveillance, rather than plaque-engulfment, potentially relevant to vascular and inflammatory components of ARIA where microglial motility and cytokine signaling are heightened while phagocytosis is reduced.
8.3.6 Cluster 5
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Itgam | Microglia | DAM/phagocytic | Downregulated | Encodes CD11b; key for adhesion and phagocytosis; reduction indicates lowered phagocytic activation. |
| Ifi207 | Microglia | Interferon-responsive/reactive | Upregulated | IFN-inducible gene; upregulation marks antiviral/innate immune activation. |
| C1qb | Microglia | Complement-mediated DAM | Downregulated | Complement component; downregulation suggests reduced synaptic pruning and opsonization. |
| Ccl4 | Microglia | Chemotactic/inflammatory | Upregulated | Pro-inflammatory chemokine; promotes immune cell recruitment to CNS lesions. |
| Lilrb4a | Microglia | Regulatory/immune-checkpoint | Upregulated | Inhibitory receptor; induced in inflammatory contexts to limit activation. |
| Ly86 | Microglia | Innate immune/TLR signaling | Upregulated | MD-1 protein; modulates TLR4/MD-2 signaling in microglial innate immune responses. |
| C1qc | Microglia | Complement-mediated DAM | Downregulated | Complement cascade subunit; loss further supports reduced opsonization activity. |
| Fgf13 | Microglia | Reactive/stress-adaptive | Upregulated | Cytosolic growth factor family member; linked to microtubule stabilization in reactive states. |
| C1qa | Microglia | Complement-mediated DAM | Downregulated | Initiator of classical complement pathway; decreased expression suggests suppressed complement activity. |
| Klf6 | Microglia | Pro-inflammatory transcriptional reprogramming | Upregulated | Induced by stress/injury; regulates inflammatory and migratory genes. |
| Dusp1 | Microglia | Anti-inflammatory/feedback | Upregulated | MAPK phosphatase; limits MAPK-driven pro-inflammatory signaling. |
| Atf3 | Microglia | Immediate-early gene (IEG)-activated | Upregulated | Stress-responsive transcription factor; marks acute activation. |
| P2ry12 | Microglia | Homeostatic | Downregulated | Purinergic receptor; loss is a hallmark of activated, non-homeostatic states. |
| Marcks | Microglia | Cytoskeletal/homeostatic | Downregulated | Membrane–actin crosslinker; reduced levels impair process motility and membrane dynamics. |
| Rgs10 | Microglia | Homeostatic/anti-inflammatory | Downregulated | Regulator of G-protein signaling; decreased levels permit stronger chemokine receptor signaling. |
| Cst3 | Microglia | Homeostatic/lysosomal | Downregulated | Cystatin C; lysosomal protease inhibitor; loss suggests altered proteolytic control. |
| Ctss | Microglia | DAM/phagocytic | Downregulated | Cathepsin S; key for antigen processing and myelin degradation; reduced expression indicates diminished phagolysosomal activity. |
| Hexb | Microglia | Homeostatic | Downregulated | β-Hexosaminidase subunit; essential for lysosomal function; decrease reflects impaired lysosomal metabolism. |
| Laptm5 | Microglia | Lysosomal/immune activation | Downregulated | Lysosomal membrane protein; reduction suggests suppressed lysosomal activity. |
| Xylt1 | Microglia | ECM remodeling/reactive | Upregulated | Glycosyltransferase in proteoglycan biosynthesis; may reflect ECM reorganization during injury response. |
Cluster 5 exhibits a marked downregulation of complement components (C1qa/b/c) and multiple lysosomal/phagocytic markers (Itgam, Ctss, Hexb, Laptm5), indicating a suppressed DAM-like phagocytic program. Concurrently, upregulated genes point to a mixed interferon-responsive and inflammatory signaling state (Ifi207, Ccl4, Ly86, Klf6, Atf3) with regulatory feedback mechanisms (Dusp1, Lilrb4a) and ECM remodeling (Xylt1). Loss of homeostatic markers (P2ry12, Marcks, Rgs10, Cst3) reinforces a non-homeostatic activated phenotype, biased toward cytokine production and immune modulation rather than debris clearance.
8.3.7 Cluster 6
| Gene | Cell | Subtype | Regulation | Notes |
|---|---|---|---|---|
| Ifit3 | Microglia | Type I interferon–responsive microglia (IRMs) | Upregulated | IFN-induced protein; hallmark of antiviral/innate immune activation in microglia. |
| Ifi211 | Microglia | IRMs | Upregulated | Interferon-inducible family member; part of broad IFN-stimulated gene (ISG) program. |
| Ifi206 | Microglia | IRMs | Upregulated | ISG; contributes to innate antiviral defense. |
| Ifit2 | Microglia | IRMs | Upregulated | Antiviral effector limiting viral replication; robustly induced by IFN signaling. |
| Rsad2 | Microglia | IRMs | Upregulated | Viperin; antiviral protein regulating lipid metabolism during innate responses. |
| Isg15 | Microglia | IRMs | Upregulated | Ubiquitin-like modifier; critical in IFN-induced protein modification during antiviral states. |
| Mx1 | Microglia | IRMs | Upregulated | Dynamin-like GTPase; classical ISG conferring antiviral resistance. |
| Ifit3b | Microglia | IRMs | Upregulated | Paralog of Ifit3; IFN-inducible, part of viral RNA sensing and translation suppression pathway. |
| Ifi213 | Microglia | IRMs | Upregulated | ISG with poorly characterized function; linked to innate immunity. |
| Oasl2 | Microglia | IRMs | Upregulated | 2′–5′-oligoadenylate synthase–like; antiviral RNA degradation pathway. |
| A330040F15Rik | Microglia | IRMs | Upregulated | Predicted IFN-inducible transcript; uncharacterized in function. |
| Ifi209 | Microglia | IRMs | Upregulated | Member of IFN-inducible family; potential nucleic acid sensor. |
| Slfn5 | Microglia | IRMs | Upregulated | Schlafen family protein; modulates immune responses and cell cycle during activation. |
| Gm4951 | Microglia | IRMs | Upregulated | Predicted IFN-inducible gene; enriched in viral defense transcriptional programs. |
| Ifitm3 | Microglia | IRMs | Upregulated | Restricts viral entry by modifying endosomal membranes. |
| Stat1 | Microglia | IRMs | Upregulated | Master transcription factor for type I/II IFN responses; drives IRM transcriptional network. |
| Ifi207 | Microglia | IRMs | Upregulated | ISG; part of IFN-driven antiviral gene set. |
| Rnf213 | Microglia | IRMs | Upregulated | E3 ligase with roles in immunity and vascular remodeling; induced by IFNs. |
| Sp100 | Microglia | IRMs | Upregulated | Nuclear body protein; upregulated in antiviral and inflammatory contexts. |
| Herc6 | Microglia | IRMs | Upregulated | E3 ligase mediating ISGylation; essential for ISG15 conjugation pathway. |
Cluster 6 shows uniform upregulation of type I interferon–stimulated genes (ISGs), including antiviral effectors (Ifit3, Rsad2, Mx1, Ifitm3), nucleic acid sensors (Oasl2, Stat1, Sp100), and ISGylation machinery (Isg15, Herc6). This profile is characteristic of interferon-responsive microglia (IRMs), a distinct activation state often triggered by viral infection, nucleic acid release from damaged cells, or chronic inflammatory cues. The absence of downregulated homeostatic or phagocytic genes suggests a strong, focused IRM polarization rather than mixed activation with DAM or AIM features.
8.3.8 Summary
| Cluster | Literature-defined subtype | Key evidence from markers | Canonical refs |
|---|---|---|---|
| 0 | Reactive/inflammatory microglia (NF‑κB/IEG‑high AIM) | Cytokines/chemokines and NF‑κB/IEG axis (Tnf, Il1b, Ccl3/4, Nfkbia, Nfkbiz, Egr1, Junb) indicating an acute inflammatory transcriptional program distinct from DAM. | Frameworks distinguishing reactive vs homeostatic/DAM microglia and nomenclature guidance. (ScienceDirect, PMC) |
| 1 | Transitional/homeostatic‑leaning (resolution/quiescent) | Broad downregulation of IEGs and inflammatory TFs (Jun, Rel, Atf3, Zfp36 family) with minimal activation markers → consistent with a resolving, low‑reactivity state along the reactive→homeostatic continuum. | Microglial state continuum and resolution concepts. (ScienceDirect, Frontiers) |
| 2 | Reactive/IEG‑activated (AIM) with preserved identity | IEG/cytokine program (Fos, Jun, Il1a, Socs3, Ccr5) but retention of homeostatic identity (Tmem119↑, Klf2↑) and non‑DAM features (Trem2/Cd68/Ctsb/Ctsd↓). | Distinction of reactive/“activated response” states from DAM and homeostatic cores. (Cell, PMC) |
| 3 | Spp1⁺ DAM with MHC‑II⁺ antigen‑presenting ARM module | Canonical DAM/MGnD markers (Spp1, Gpnmb, Itgax, Cst7, Lgals3) with suppression of homeostatic genes (P2ry12↓), plus strong MHC‑II antigen‑presentation (H2‑Aa/Ab1/Eb1, Cd74). | DAM/MGnD (TREM2–APOE axis) and ARM/antigen‑presenting programs. (ScienceDirect, PMC, Cell) |
| 4 | Chemotactic/migratory reactive microglia (non‑DAM) | Motility/chemotaxis signaling (Ccr5, Rapgef5, Cacnb2, Frmd4a) retained identity (Tmem119↑) with phagolysosomal/DAM axis down (Trem2, Tyrobp, Cd68, Ctsb/Ctsz, Lyz2↓) → reactive but non‑phagocytic. | Reactive (non‑DAM) states within current nomenclature; distinctions from DAM signature. (ScienceDirect, Cell) |
| 5 | Cytokine/immune‑modulatory reactive microglia with complement/lysosome‑low | IFN‑tinted inflammatory program (Ifi207, Atf3, Klf6, Ccl4 ↑) alongside reduced complement/lysosomal/DAM machinery (C1qa/b/c, Ctss, Hexb, Laptm5 ↓) and loss of homeostatic markers (P2ry12↓). | Reviews summarizing non‑DAM reactive states vs DAM/complement modules. (ScienceDirect, Alzheimer’s Journals) |
| 6 | Interferon‑responsive microglia (IRM) | Uniform ISG induction (Ifit2/3/3b, Isg15, Mx1, Rsad2, Oasl2, Stat1, Herc6) defining the IFN‑I program in microglia. | IRM definition and markers in mouse brain. (PMC, Cell) |
Across clusters, we observe classic Spp1⁺ DAM/ARM features (cluster 3), a robust IFN‑I IRM state (cluster 6), and multiple reactive, non‑DAM states (clusters 0, 2, 4, 5) differing by IEG/NF‑κB intensity, chemotaxis/motility bias, and complement/lysosome suppression. One cluster trends toward resolution/quiescence (cluster 1). Together, these assignments align with established microglial taxonomies distinguishing homeostatic, reactive (AIM/ARM), DAM/MGnD, and IRM states in mouse models of neurodegeneration and inflammation. (ScienceDirect, PMC, Cell)